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General

Gibco™ Geltrex™ Matrix and collagen (rat tail and bovine) can be used as either a coating solution or a 3D gel matrix. CELLstart™ Substrate was developed to be used as a xeno-free coating matrix for only ESC applications (as a substitute for Gibco™ Geltrex™ Matrix or Matrigel™ Matrix, which are of animal origin). AlgiMatrix™ Matrix is a 3D scaffold–type matrix that does not support cell attachment but does provide a good environment for growing spheroids that can be easily harvested for downstream applications.

Gibco™ Geltrex™ Matrix

Gibco™ Geltrex™ Matrix is a soluble form of reduced growth factor (RGF) basement membrane extract (BME) purified from murine Engelbreth-Holm-Swarm tumor. Historically these preparations have contained the lactate dehydrogenase-elevating virus (LDEV), which does not impact the performance of the material but may be of concern in certain applications such as xenografts or other clinically focused research. New manufacturing methods are now available that allow for the removal of this virus. The resulting LDEV-free material is tested using PCR methods to confirm that the material is virus-free. 

Gibco™ Geltrex™ Matrix is a basement membrane extract that contains collagen IV, laminin, and other matrix proteins and growth factors, which are required for cell adhesion. AlgiMatrix™ Matrix contains only alginate, which does not facilitate cell adhesion. There is nothing in the alginate sponges for the cells to interact with.

Yes, try a variety of dilutions to coat the plates. Some customers use a 1:100 dilution and have better results. The optimal dilution is cell-line dependent.

If you would like to culture the cells once recovered from the matrix, we recommend washing the cells first, then using trypsin/EDTA, TrypLE™ reagent, or a solution of 3,000 U/mL collagenase and 1,000 U hyaluronidase in low-glucose DMEM. If you would like to monitor cell surface markers, we recommend removal of the cells by mechanical means, such as a vigorous wash or a delicate scrape. 

Note that the phenotype of the cells may change if they are cultured in 2D after their removal from the matrix.

Lactate dehydrogenase-elevating virus (LDEV) infects mouse macrophages and induces persistent infections in laboratory mice. LDEV, an RNA virus, alters in vivo physiology, and is a common contaminant of biological materials derived from transplantable tumor cell lines.

Yes, this is normal. It has been observed in product development. Remove the Gibco™ Geltrex™ Matrix solution after pre-coating and add cells as normal. H9 human embryonic stem cells have been cultured on dishes pre-coated with Ready-to-Use Gibco™ Geltrex™ Matrix with this observation in the pre-coating procedure. 

Ready-to-Use Gibco™ Geltrex™ Matrix provides only a coating for cell attachment and was developed mainly for ESC and iPSC use. Because the protein concentration is not high enough, it should not be used for thick gel or 3D applications.

AlgiMatrix™ 3D Culture System

AlgiMatrix™ Matrix is stable for 12 months unopened. Once opened, the entire plate needs to be used.

We have shown the following data:

  1. Focusing on cells in culture shows evidence of inoculation.
  2. Paraffin embedding and sectioning shows that cells are inoculated throughout the sponge.

Add medium gently and at an angle and gently. Be careful not to impel the matrix when re-feeding.

Spheroid formation requires about 5 to 7 days once inoculated. Air bubbles will be observed at the time of inoculation. This will dissipate within several days by the cells consuming the oxygen.

The Invitrogen™ alamarBlue™ reagent assay is a useful biological assay that monitors the health and function of cells. 

Yes, start by dissolving the matrix using calcium binding solution, Versene, or sodium citrate. The remaining spheroids can be dissociated with TrypLE™ reagent, counted, and expanded into other culture vessels or into new AlgiMatrix™ Matrix sponges. These steps are illustrated in the instruction manual that is supplied with each kit.

While we have not tested this in-house, the common belief is that if the alginate scaffolds support proliferation of human ES without feeder cells, they should also support mouse ES. Both mouse and human form embryoid bodies, and seeding within the macro-porous alginate scaffolds have been found very advantageous for these structures, in terms of proliferation and differentiation.

There is virtually no background staining with alginate. The same surface characteristic that prevents cells from adhering also makes it very difficult for molecules such as fluoresceinated antibodies to adhere, thus essentially no background staining is observed.

This scaffold is great for forming 3D spheroids that allow maximum cell to cell interaction. Established cell lines expand by increasing size of spheroids up to ~100 cells or so depending upon the cell type. Both primary and established cells will differentiate in a 3-dimensional format over 1–2 weeks of culture. 

For umbilical cord stem cells, you might want to try a relatively high inoculation density of 100,000–300,000 cells per sponge with daily or every-other-day re-feeding of cultures, as the medium turns "yellow".

Standard immunofluorescence testing can be performed on 3D structures. The staining can be done with either intact spheroids within the AlgiMatrix™ Matrix or you can dissolve the AlgiMatrix™ Matrix using Versene or 55 mM tri-sodium citrate dihydrate and stain the spheroid alone. You can further dissociate the spheroid into individual cells using TrypLE™ reagent and stain as dissociated cells. 

Invasion studies measure the ability of cells to penetrate through a matrix, simulating a metastatic cells activity in vivo. AlgiMatrix™ Matrix contains only alginate (brown seaweed) with no in vivo-like molecules in the undecorated structure, so invasion may not be a very relevant use for AlgiMatrix™ Matrix. 

Our supplier has been producing pharmaceutical-grade alginate and hyaluroinic acid for years. Although these are natural products, by specifying among other things (in the case of alginate) harvest location, water temperature, which part of the seaweed to harvest, use of stringent purification SOPs, and rigorous QC criteria, a very standard product can be obtained.

The recent more highly purified forms of alginate do not generate a sensitivity reaction as is the case with less pure versions. Alginate has been used for some years to encapsulate, for instance, B-cells of the pancreas for in vivo transplantation (diabetes research) and it does not get rejected. 

Alginate, from which AlgiMatrix™ Matrix is made, presents a very hydrophilic surface, and lipid-containing cell membranes and even molecules do not adhere. Normal cells will aggregate to form spheroids and differentiate into structures in AlgiMatrix™ Matrix, but expansion will be limited since most normal cells need to adhere to increase in number. Cancer cells are able to expand without attachment and do so in AlgiMatrix™ Matrix (one exception to this is embryonic stem cells, which can expand as embryoid bodies in AlgiMatrix™ Matrix). However, here is one major difference between soft-agar and AlgiMatrix™ Matrix: soft-agar is a hydrogel and AlgiMatrix™ Matrix is a sponge with relatively large pore sizes, and cells that do grow will be able to do so easier and faster in AlgiMatrix™ Matrix than when encased in a hydrogel (soft-agar). So a traditional way to tell cancer cells from normal cells is to observe the culture for expansion; cancer cells (example HepG2 hepatocarcinoma) will form expanding spheroids while normal cells will form spheroids that do not increase in size or number.

The height of the sponge is ~3–4 mm dry. When the sponge is hydrated, its volume is essentially the same— it doesn't swell and increase in size, in fact it becomes slightly bit smaller as it essentially goes back into a hydrogel consistency, especially if the inoculated sponges are centrifuged briefly (as suggested to force cells more into the interior and also to spread the sponge uniformly over the bottom of the well).

Some primary cells (with the exception of stem cells) need to adhere in order to expand. As cells won’t adhere to AlgiMatrix™ Matrix, these cells can still be cultured, but will tend to differentiate rather than expand. We recommend that you plate at a high cell density for primary cells.

3D cell culture with AlgiMatrix™ Matrix approaches a more physiological state in terms of cell growth and behavior, but along with that comes the higher variability typically associated with more complex systems. You may need to do more replicates or more controls.

The firming buffer works by adding calcium to the sponge, making it stiffer. The dissolving buffer works by removing calcium from the sponge, making it softer. 

Other than the Invitrogen™ alamarBlue™ Cell Viability Reagent, AlgiMatrix™ Matrix is also compatible with Invitrogen™ PrestoBlue™ Cell Viability Reagent, Promega™ Cell Titer-Glo™ reagent, or Invitrogen™ CyQuant™ Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix™ Matrix as a blank control. 

Collagen I

The key applications include the following:

  • Collagen I induces microvascular endothelial cells to adopt a spindle-shaped morphology, in vitro, and to align into solid cord-like assemblies. Vascular endothelial cells can also form vessel-like, tubular structures when cultured on collagen I. Collagen I can be used for in vitro angiogenesis assays.
  • Breast cancer stem cells can undergo differentiation when cultured on collagen I.
  • Collagen I has been used for the culture of primary colon carcinoma cell lines; mouse liver progenitor cells have been cultured in 3D collagen I and rat pancreatic islets.
  • Collagen I can act as a barrier in cell invasion assays, and has been used to study cell adhesion.

The following cell types are commonly cultured in collagen I:

  • Endothelial cells (including HUVEC)
  • Fibroblasts
  • Hepatocytes
  • Chondrocytes
  • Osteoclasts
  • Osteoblasts
  • Keratinocytes
  • Corneal epithelium

Enzymes that can be used include: Gibco™ TrypLE™ Express Enzyme, Gibco™ TrypLE™ Select Enzyme, or Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent.

Collagen I is provided as a liquid, suspended in 20 mM acetic acid solution. If difficulties are encountered with the preparation, the collagen can be diluted in 1:2, first in 20 mM acetic acid, pH 3.5 before further diluting according to the protocol being used.

Recombinant Human Laminin-521 (rhLaminin-521)

Yes. To ensure optimum recovery of PSCs following single-cell passaging, PSCs should be fed with Essential 8™ Flex Medium the day before passaging.

The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 μg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 μg/cm2.

We recommend that you leave the product on ice while prepping to use it.

Yes, you may use EDTA or versene. However, we do not recommend using dispase or collagenase as it can lead to differentiation.